The Lin‐11, Isl‐1, and Mec‐3 domains (LIM) transcription factors play essential roles in regulating plant biological processes. Despite that, there is a lack of a full understanding of LIMs in wheat (Triticum aestivum L.). In this study, 28 wheat LIM s (TaLIMs) were identified and designated as TaLIM1‐1A to TaLIM12‐7D. The cis‐regulatory element analysis showed that TaLIMs were rich in elements related to biological and abiotic stresses. Expression profiling analysis showed that certain members of TaLIMs were responsive to biotic and abiotic stresses, such as TaLIM1‐1A, TaLIM3‐2B, TaLIM8‐4D, and TaLIM10‐5D, were significantly induced by heat, drought, sodium chloride (NaCl), abscisic acid (ABA) and Fusarium graminearum stresses. Furthermore, the biological function of TaLIM8‐4D was analyzed and results showed that it was subcellular localization in the nucleus and could induce weak cell death in Nicotiana benthamiana leaves. Additionally, overexpression of TaLIM8‐4D could upregulate plant pathogenesis‐related (PR) genes, promoting the infection of hemibiotrophic pathogen, implying that TaLIM8‐4D could function as susceptible gene in the nucleus by upregulating PR genes and inducing cell death to promote the colonization of hemibiotrophic agent F. graminearum. Overall, the systematic identification, characterization, expression profiling, evolutionary, and function analyses provided the ability to understand TaLIMs and laid a foundation for the further function study of LIM family members in wheat.

Background Plant plasma membrane transporters play essential roles during the translocation of vectorized agrochemicals. Therefore, transporters associated with phloem loading of vectorized agrochemicals have drawn increasing attention. As a model system, castor bean (Ricinus communis L.) has been widely used to detect the phloem mobility of agrochemicals. However, there is still a lack of an efficient protocol for the Ricinus seedling model system that can be directly used to investigate the recognition and phloem loading functions of plasmalemma transporters toward vectorized agrochemicals. Results Here, using vacuum infiltration strategy, we overexpressed the coding gene for enhanced green fluorescent protein (eGFP) in R. communis seedlings by Agrobacterium tumefaciens-mediated transformation system. Strong fluorescence signals were observed in leaves, demonstrating that exogenous genes can be successfully overexpressed in seedlings. Subsequently, gene expression time and vacuum infiltration parameters were optimized. Observation of fluorescence and qRT-PCR analysis showed that eGFP strength and expression level reached a peak at 72 h after overexpression in seedlings. Parameter optimization showed Agrobacterium concentration at OD600 = 1.2, and infiltration for 20 min (0.09 MPa), return to atmospheric pressure, and then infiltration for another 20 min, were the suitable transformation conditions. To test the application of vacuum agroinfiltration in directly examining the loading functions of plasma membrane transporters to vectorized agrochemicals in seedlings, two LHT (lysine/histidine transporter) genes, RcLHT1 and RcLHT7, were overexpressed. Subcellular localization showed the strong fluorescent signals of the fusion proteins RcLHT1-eGFP and RcLHT7-eGFP were observed on the cell membrane of mesophyll cells, and their relative expression levels determined by qRT-PCR were up-regulated 47- and 52-fold, respectively. Furthermore, the concentrations of l-Val-PCA (l-valine-phenazine-1-carboxylic acid conjugate) in phloem sap collected from seedling sieve tubes were significantly increased 1.9- and 2.3-fold after overexpression of RcLHT1 and RcLHT7, respectively, implying their roles in recognition and phloem loading of l-Val-PCA. Conclusions We successfully constructed a transient expression system in Ricinus seedlings and laid the foundation for researchers to directly investigate the loading functions of plasma membrane transporters to vectorized agrochemicals in the Ricinus system.

Amino acid conjugates of pesticides can promote the phloem translocation of parent ingredients, allowing for the reduction of usage, and decreased environmental pollution. Plant transporters play important roles in the uptake and phloem translocation of such amino acid-pesticide conjugates such as L-Val-PCA (L-valine-phenazine-1-carboxylic acid conjugate). However, the effects of an amino acid permease, RcAAP1, on the uptake and phloem mobility of L-Val-PCA are still unclear. Here, the relative expression levels of RcAAP1 were found to be up-regulated 2.7-fold and 2.2-fold by the qRT-PCR after L-Val-PCA treatments of Ricinus cotyledons for 1 h and 3 h, respectively. Subsequently, expression of RcAAP1 in yeast cells increased the L-Val-PCA uptake (0.36 μmol/107 cells), which was 2.1-fold higher than the control (0.17 μmol/107 cells). Pfam analysis suggested RcAAP1 with its 11 transmembrane domains belongs to the amino acid transporter family. Phylogenetic analysis found RcAAP1 to be strongly similar to AAP3 in nine other species. Subcellular localization showed that fusion RcAAP1-eGFP proteins were observed in the plasma membrane of mesophyll cells and phloem cells. Furthermore, overexpression of RcAAP1 for 72 h significantly increased the phloem mobility of L-Val-PCA in Ricinus seedlings, and phloem sap concentration of the conjugate was 1.8-fold higher than the control. Our study suggested that RcAAP1 as carrier was involved in the uptake and phloem translocation of L-Val-PCA, which could lay foundation for the utilization of amino acids and further development of vectorized agrochemicals.

Background: The LIM (Lin-11, Isl-1 and Mec-3 domains) transcription factors play essential roles in regulating plant biological processes, including cytoskeletal organization, development of secondary cell wall, and cell differentiation. Despite their important roles, there lacks a fully understanding of LIMs in wheat.Results: In this study, 28 TaLIMs (Triticum aestivum LIM) were identified and designated as TaLIM1-1A to TaLIM12-7D. Phylogenetic relationship analysis showed that TaLIMs could be divided into two groups (Group Ⅰ and Group Ⅱ), and the exon/intron structure analysis suggested that members in the same group have similar gene structure. Chromosome mapping showed that 28 TaLIMs were unevenly distributed on 18 chromosomes. Motif analysis revealed that 28 TaLIMs contained 15 motifs, of which motif1 (LIM-domain) was detected in all TaLIMs. Protein characterization indicated that TaLIMs were hydrophilic proteins and most of them were unstable. They were mainly comprised by α-helix and β-turn and without signal peptides. Duplication, Ka/Ks, and synthesis analyses suggested that polyploidization played fundamental role in the expansion of TaLIM members. Function annotations indicated that all TaLIMs were annotated under GO terms “zinc ion binding” (GO:0008270). The cis-regulatory element analysis showed that TaLIMs were rich in elements related to biological/abiotic stress, growth and development, and phytohormone response. RNA-seq data analysis showed certain members of TaLIMs were responsive to biotic and/or abiotic stresses. Such as TaLIM1-1A, TaLIM3-2B, TaLIM8-4D, and TaLIM10-5D were significantly induced by heat, drought, NaCl, ABA and Fusarium graminearum stresses. The subcellular localization of TaLIM8-4D show that it was in the nucleus. Furthermore, the biological function of TaLIM8-4D was analyzed and results showed that it could induce weak cell death in Nicotiana benthamiana leaf. Besides, overexpression of TaLIM8-4D could up-regulate plant pathogenesis-related (PR) genes, whereas promoted the infection of hemi-biotrophic pathogen, implying that TaLIM8-4D could function as susceptible gene in nucleus by up-regulating PR genes and inducing cell death to promote the colonization of hemi-biotrophic agent F. graminearum.Conclusion: The systematic identification, characterization, expression profiling, evolutionary, and function analyses provided us the fully understanding to TaLIMs and laid a foundation for the further function study of LIM family members in wheat.